Title:RNF170Regulates Cell Apoptosis And Autophagy In Human Lung Cancer Cells

ObjectiveE3ligase plays an important role in the process of ubiquitin proteasome degradation. However, the substrates and function of many E3ligases are still unknown at present. Recent research indicates that E3ligase could impact several vital biological processes including autophagy. RNF170is an RING family E3ligase. RNF170could medidate the ubiquitination and degradation of IP3R, which is the Ca2+channel protein. And there’s little additional research of RNF170. Here in our research, we studied the function of RNF170in cell autophagy and apoptosis, and further researched the mechanism:RNF170inhibited autophagy through mTOR pathway. We further completed the function of RNF170.Methods1. Cell culture.2. Autophagosome fluorescence assay was used to measure cell autophagy.3. The changes of proteins were examined by Western Blot.4. Apoptosis was measured by Flow Cytometry.5. Knockdown of target gene was achieved by transfection with specific siRNA.6.Overexpression of target protein was achieved by transfection with plasmid.7.Immunoprecipitation was used to measure protein interaction. Results1. After the induction of cell autophagy with different methods in non-small cell lung cancer cell line A549, the level of RNF170expression decreased; Using different concentration of Salermide treated A549and H1792, RNF170expression decreased while LC3B increased, and the change was concentration-dependent. 2. In glioma cell line U87-MG-LC3-GFP which steady expressed LC3-GFP, autophagosome fluorescence increased after RNF170knockdown.3. RNF170knockdown triggered cell autophagy in A549, H1792and Calu-1with different drug treated.4. Overexpression of RNF170decreased cell autophagy in H1792cell line.5. After RNF170knockdown in A549, H1792and Calu-1, the expression of pp70S6K and p-4EBP1decreased, which were the downstream of m-TOR pathway; When RNF170was overexpressed in H1792, the level of pp70S6K and p-4EBP1increased obviously.6. RNF170knockdown increased cell apoptosis in A549and H1792, flow cytometry also detected increased cell apoptosis level.7. IP3R played an important role during the regulation of autophagy and apoptosis of RNF170.8. ATG3and RNF170may have an interaction with immunoprecipitation proof.Conclusions1. RNF170impacted cell autophagy, RNF170decreased when autophagy happened.2. RNF170inhibited cell autophagy, the level of cell autophagy rised when RNF170knockdown and reduced when RNF170was overexpressed.3. RNF170regulated autophagy through the downstream of m-TOR(the expression of pp70S6K and p-4EBP1).4. RNF170inhibited cell apoptosis.5. RNF170may regulate cell autophagy and apoptosis through IP3R and ATG3. In summary, we found E3ligase RNF170can regulate cell autophagy and apoptosis in non-small cell lung cancer cells, and further explained the molecular mechanism. RNF170could decreased the level of LC3B through pp70S6K and p-4EBP1, which were the downstream of m-TOR pathway. RNF170can also inhibit cell apoptosis, the level of apoptosis increased after RNF170konckdown. RNF170may regulate cell autophagy and apoptosis through IP3R and ATG3.